What about proteins allows them to be separated?
Physical properties
How are antibodies useful in identifying proteins?
Protein identification and purification using antibodies
Homoginization
any of several processes used to make a mixture of two mutually non-soluble liquids the same throughout. This is achieved by turning one of the liquids into a state consisting of extremely small particles distributed uniformly throughout the other liquid
First step in protein purfication
centrifugation
What is the purpose of homogenization in protein purification?
organelles and cytoplasmic proteins are released into solution
What is the purpose of low force differential centrifugation?
Separate organelles that have various masses that permit them to be separated
Why do we want to separate organelles via centrifugation?
To be able to isolate proteins from specific organelles or the cytoplasm, which can be further purified
Centrifugal differentiation
use of different centrifugal forces to further separate things based on their mass
What are the different protein purification techniques?
Salting out
Centrifugation
Dialysis
Gel filtration chromatography
Ion-exchange chromatography
Affinity chromatography
High-Pressure Liquid chromatography
Salting out
separating proteins by their respective solubilities (charge)
Gel Filtration Chromatography
separating proteins by their size (shape)
Ion-exchange Chromatography
separating proteins by their charge
Affinity Chromatography
purifying proteins based on different surface features or ligand binding properties
High Pressure Liquid Chromatography
high resolution method for purifying proteins
ammonium sulfate (NH4)SO4
commonly used to precipitate proteins
What determines when proteins precipitate?
Precipitate at different concentrations depending on the protein's respective solubilities
Centrifugation
used to isolate precipitated proteins
Dialysis
remove salt or residual ammonium sulfate associated with the protein
Process of dialysis
after centrifugation. precipitated proteins are resuspended in buffer and placed in a dialysis bag
membrane of the dialysis bag has small pores that allow ions and small molecules to diffuse out of the dialysis bag while larger molecules are retained in t
What component of proteins is used to separate proteins in gel-filtration chromatography?
Size of proteins
What component of gel filtration makes it possible to separate the proteins by size?
very small beads
What size species will elute first in gel filtration?
larger will elute first
Why do larger species elute first in gel filtration?
They either are too large to enter the pores or they can only enter a few - spend less time in the bead's pores
Knowing proteins consist of a known size, what important information can be estimated?
Approximate molecular weight
In ion-exchange chromatography, what characteristic are proteins separated upon?
charge
Assuming the beads in ion-exchange are negative, which species will elute off?
negative species - repelled from negative beads
What is used to get positively charged proteins off of negatively charged beads?
gradient of increasing salt concentration
Ex. NaCl
This is because salt competes with protein for binding to the beads
How could one purify negatively charged proteins?
on an ion-exchange column of positively charged beads
The charge of the protein is strongly influenced by the ____
pH of the buffer
What is a ligand?
substance that forms a complex with a biomolecule to serve a biological purpose. In protein-ligand binding, the ligand is usually a signal-triggering molecule binding to a site on a target protein. The binding typically results in a change of conformation
Affininty chromatography
separation based on surface features or ligand binding.
Small molecules are attached to beads and a mixture of protein is applied. Protein that binds the small molecule is retained on the column, while all other protein are washed off the column. Bound pr
Which type of chromatography can yield protein purity >95% in a single step?
affininty chromatography
Examples of affinity chromatography
metal-protein
protein-ligand
protein-protein
enzyme-substrate
enzyme-inhibitor
antibody-protein
Why is high pressure required for HPLC?
Columns are able to have smaller, more finely divided beads
Why is it good to use such finely divided beads in HPLC?
superior resolving power, high separation of proteins
Types of gel-electrophoresis
PAGE - polyacrylamide Gel Electrophoresis
SDS-PAGE
Isoelectric Focusing
2D Gel-Electrophoresis
Polyacrylamide Gel Electrophoresis (PAGE)
molecules are separated by size and charge in an electric field through porous gel matrix
Which molecules travel fastest in PAGE
Smaller molecules
What is the difference between proteins separated though PAGE and SDS-PAGE?
In regular PAGE, proteins are in their natural state (folded and disulfides intact)
In SDS PAGE, proteins denatured, disulfide bonds reduced
SDS-PAGE
Electrophoresis under denaturing conditions
What is SDS?
Sodium dodecyl sulfate
negatively charged deterrent that denatures proteins
What is beta-mercaptoethanol?
reduces disulfide bonds in proteins to be separated in SDS-PAGE
Why do all proteins in SDS PAGE travel toward the positively charged terminal?
All proteins are coated with a negative detergent
What information can we learn from proteins based on their speed of travel towards the positive terminal in SDS PAGE?
They travel at a speed inversely proportional to their size (number residues)
Describe the charge-to-mass ratio of proteins in SDS PAGE
all have similar q/m ratio
How can one visualize proteins in SDS PAGE?
Must be stained with a dye, like Coomassie Blue
Isoelectric Focusing
proteins are loaded onto a gel with a gradient of low to high pH
voltage applied to gel
proteins migrate to where they have a net charge of 0 (isoelectric point or pl value of the protein)
Why do proteins stop moving in isoelectric focusing?
They are no longer influenced by the gel when they reach the desired pH and they stop moving
What is the isoelectric point or pI value of a protein?
the pH at which a protein no longer moves in isoelectric focusing
Two-Dimensional Gel Electrophoresis
proteins separated in two directions with 2 techniques
1. Proteins separated with isoelectric focusing (by charge)
2. SDS-PAGE and separate by molecular weigh
- individual proteins can be analyzed by mass spectrometry
Ways of using antibodies to purify and characterize proteins
protein immunoblotting (western)
protein purification by immunoprecipitation
Immunoglibin G
antibody
quatenary structure consists of 4 chains: 2 heavy and 2 light chains - liked with disulfide bonds
Where is the antigen binding site?
Fab domain
What does the antigen binding site do?
binds to specific antigen - could be carbohydrates, nucleic acids, proteins
Polyclonal
mixture of antibodies that recognize different epitopes
Monoclonal
Single type of antibody isolated from an antibody-producing cell called a hybridoma
Homogenous- recognizes one specific epitope
Epitope
part of the antigen that is recognized
Pros of using antibodies to detect proteins
Antibodies have high affinities and specificities for proteins
What is a western blot used for?
detecting a specific protein on a gel
How are proteins transferred to a polymer sheet from the gel?
via an electric current
SDS-covered proteins travel to positive terminal on other side of the page
Steps of a western blot (aka immunoblotting)
1. Separate proteins of interest on an SDS-PAGE gel
2. Transfer the proteins from the gel onto a polymer sheet via an electric current
3. Polymer sheet exposed to antibody specific for the protein of interest
4. Position of antibody detected with a second
In western blotting, does the secondary antibody bind to the protein or the primary antibody?
primary antibody - 2 antibodies stacked
Steps of Immunoprecipitation
1. Beads that are linked covanlently to an antibody specific for the estrogen receptor are added to a cytoplasmic fraction containing the estrogen receptor
2. Estrogen receptor binds to the antibody-linked beads
3. Sample is centrifuged and washed to remo
cytosolic
cytoplasmic
Methods used for analyzing protein sequences and structures
Protein sequencing
- Edman Degradation
- DNA sequencing
Mass Spectrometry - accurate determination of masses of proteins and peptides
Three-dimensional Structures of Proteins and Nucleic Acids
- determined by X-ray crystallography and Nuclear Magnetic Res
Back in the day, how did scientists sequence proteins? (2)
complete acid hydrolysis of peptide bonds
N-terminally sequenced with chem technique Edman degradation
Why is protein sequencing important?
amino acid sequence is key to understanding proteins functions
Today, how are proteins identified?
mass spectrometry - larger proteins are cleaved into smaller peptides via chemical or enzymatic cleavage
Today, how are protein sequences found?
deduced through DNA sequencing
Mass spectrometry
measures the mass/charge ratio of ionized proteins and peptides, provides highly accurate masses and identification
When is the mass/charge ratio equal to the mass of the protein?
When the charge of the protein is equal to +1
Ex. Insulin and beta-lactoglobulin
What is different about analyzing large proteins via mass spectroscopy?
large proteins can be cleaved by proteases and the resulting peptide fragments can be analyzed by mass spectrometry
proteases
enzymes that hydrolyze proteins
What information do the peptide fragments give us when using mass spectrometry?
can be used to identify a protein and determine its amino acid sequence
X-ray crystallography
x-ray diffraction from protein crystal can determine he protein structure
What is resolution in x-ray crystallography?
determines the level of atomic detail that can be seen
(lower resolution means more detail)
What is the resolution needed to see hydrogen atoms?
1.0 � or higher
How are hydrogen bonds determined in x-ray structures?
inferred based on distance and geometry between hydrogen bond donors and acceptors
Why are crystals used to determine their structure in x-ray crystallography?
crystals represent an overall average of all of the proteins within a cell
NMR - nuclear magnetic resonance spectroscopy
can be used to determine the structures of proteins, DNA, RNA
What does NMR offer that X-ray crystallography cannot do?
provide information about the dynamics of proteins and nucleic acids and also gives info about H atoms and interactions
What are the uncertainties associated with NMR? What is done about this?
there can be multiple models for protein's three-dimensional structure.
The multiple models are reported
What would be the advantages of using X-ray crystallography over NMR?
NMR relies upon molecular mechanics to determine protein or nuc acid model, lower level of experimentally determined detail than higher resolution X-ray crystal structure